Conversion of acid S, produced by the organism Polyangium cellulosum var. fulvum into acid F

ABSTRACT

A process for the conversion of the major antifungal antibiotic, acid S (ATCC No. 25532) isolated from the fermentation of Polyangium cellulosum var. fulvum into the minor antibiotic, acid F, from the same fermentation is described wherein acid S is methylated with diazomethane to provide acid S methyl ester which is then oxidized with silver carbonate on celite to obtain the corresponding keto ester S, which is subsequently reduced with sodium borohydride to give a mixture of acid S methyl ester and acid F methyl ester. These esters are readily separated by preparative thin layer chromatography and the acid F methyl ester is hydrolyzed with sodium hydroxide solution to provide acid F.

The present invention is concerned with a process for the conversion ofthe major antifungal antibiotic (acid S) isolated from the fermentationof Polyangium cellulosum var. fulvum into the minor antibiotic (acid F)from the same fermentation. Acid F is isolated either in small amountsor in many cases not at all from the fermentation medium. Thus, in orderto have an adequate supply of acid F, it has been found necessary todesign a process for the conversion of the readily available acid S intoacid F.

Acid S and acid F are described in U.S. Pat. Nos. 3,651,216 and3,804,948. As disclosed in these patents, both acid S and acid F arepotent antifungal agents. In addition, U.S. Pat. No. 3,804,948 describesthe chemical preparation of the methyl ester of acid S. Keto ester S, aderivative of acid S, disclosed in U.S. Pat. No. 3,932,620 is also usedin the process of this invention.

The process of the invention is shown in Scheme A as follows: ##STR1##Acid S (I) is methylated with diazomethane to provide acid S methylester II, which is oxidized with silver carbonate on celite to obtainketo ester S (III). Attempts to oxidize acid S directly to keto S yieldstars, but selective oxidation of acid S methyl ester to keto ester S,following the procedure described in U.S. Pat. No. 3,932,620 issuccessful.

Keto ester S(III), in an alcoholic solvent such as methanol, is reducedwith an alkali metal borohydride, typically sodium borohydride, toprovide a mixture of acid S methyl ester II and acid F methyl ester IV.The reduction step may be conducted under nitrogen. These esters arereadily separated by preparative thin layer chromatography. A typicalsolvent system which may be used for this separation is ethylacetate-cyclohexane in a ratio of 4:1. Variations of this solventsystem, commonly used in thin layer chromatography separations are alsosuitable.

The acid F methyl ester IV thus obtained is hydrolyzed to obtain acid F(V). The hydrolysis is conducted in an alcoholic solvent, such asmethanol, using alkali metal hydroxide, typically sodium hydroxide. Thehydrolysis step may be conducted under nitrogen.

To confirm the structure of acid F (V), the diacetate VI of thesemi-synthetic product was prepared. A sample of natural acid F (VII)obtained from the fermentation was also acetylated to obtain acid Fdiacetate VIII. The NMR spectra of the two diacetates VI and VIII werefound to be identical in all respects, indicating that thesemi-synthetic product VI is acid F diacetate. Thus, the semi-syntheticproduct V is identical to natural acid F (VII) isolated from thefermentation of Polyangium cellulosum var. fulvum. The infra-red spectraof semi-synthetic acid F (V) and natural acid F (VII) are alsoidentical.

The following examples are provided to further illustrate the inventionand are not be construed as limiting the scope of the invention.

EXAMPLE 1 ##STR2## Acid S Methyl Ester and Acid F Methyl Ester Sodiumborohydride (230 mg, 0.00575) is added to a solution of keto ester S(230 mg, 0.00047 mole) in methanol (35 ml). The reaction mixture isstirred under nitrogen for 2 hours. The solvent is evaporated underreduced pressure to give a white gummy solid. The solid is dissolved inwater, acidified with 1 N hydrochloric acid and extracted withchloroform. The extracts are dried over MgSO₄ and evaporated to give apale yellow gum (187 mg). The gum is fractionated into two products bypreparative TLC with the solvent system ethyl acetate-cyclohexane (4:1).Acid S Methyl Ester (most polar compound) is isolated as a colorless oil(86 mg., 37%), Acid F Methyl Ester (least polar compound) is isolated asa colorless oil (49 mg., 21%). Diagnostic TLC indicates both products tobe homogeneous and both have the same Rf values as the correspondingesters derived from the natural acids. EXAMPLE 2 ##STR3## Acid F(Semi-synthetic) 1 N sodium hydroxide solution (3 ml) is added to amethanol solution of semi-synthetic acid F methyl ester (49 mg., 0.00001mole). The reaction mixture is stirred at 90 under nitrogen for 30minutes. The methanol is removed under pressure. The aqueous residue isacidified with 1 N hydrochloric acid and extracted with chloroform. Theextracts are dried over magnesium sulfate and evaporated to give acid Fas a colorless oil (25 mg., 53%).

IR γ max broad 3600-3200 and 2800-2500 (OH), 1720 cm⁻¹ (CO)

    ______________________________________                                        .Iadd.Mass Spectrum                                                           ______________________________________                                        Observed molecular ion 474.3010                                               Calculated for C.sub.28 H.sub.42 O.sub.5                                                              474.2981                                              ______________________________________                                        .Iaddend.

m/e (relative intensity) 474(14), 456(9), 445(29), 379(19), 361(8),279(64), 245(19), 235(21), 195(75) and 193(100).

EXAMPLE 3 ##STR4## Acid F Diacetate (Semi-synthetic) Acetic anhydride (1ml) is added to a solution of semi-synthetic acid F (25 mg) in pyridine(2 ml). The solution is allowed to stand at room temperature overnight,diluted with water, and evaporated to give acid F diacetate as a paleyellow gum (28 mg 90%).

IR γ max broad 3500-3100 and 2800-2400(OH), 1745 (CO) and 1720 cm⁻¹ (CO)

    ______________________________________                                        .Iadd.Mass Spectrum                                                           ______________________________________                                        Observed molecular ion 558.3295                                               Calculated for C.sub.32 H.sub.46 O.sub.8                                                              558.3271                                              ______________________________________                                        .Iaddend.

m/e (relative intensity) 558(17), 529(50), 463(21), 343(10), 305(10),259(7), 245(23), 195(23) and 193(100).

NMR (CDCl₃) δ 0.89 (s, 3H, CH₃), 1.05 (m, 6H, 2CH₃), 1.59 (s, 3H, CH₃),1.64 (s, 3H, CH₃), 1.98 (s, 3H, CH₃ CO), 2.14 (s, 3H, CH₃ CO), 2.44 (q,1H, CH₂ CO), 2.65 (q, 1H, CH₂ CO), 3.07 (m, 1H, bisallyl), 3.86 (q, 1H,CH--O), 4.07-4.29 (m, 3H, 3CH--O), 4.67 (q, 1H, CH--OAc), 5.06 (q, 1H,vinyl), 5.25 (d, 1H, vinyl), 5.32-5.52 (m, 4H, 3 vinyl and 1 CH--OAc)and 5.57 (d, 1H, vinyl).

EXAMPLE 4 ##STR5## Acid F Diacetate (From natural product) Aceticanhydride (1 ml) is added to a solution of acid F (100 mg) in pyridine(2 ml). The solution is allowed to stand at room temperature overnight,diluted with water, and evaporated to give acid F diacetate as a lightbrown gum.

IR γ max broad 3500-3100 and 2800-2400(OH), 1745(CO) and 1720 cm⁻¹ (CO).

    ______________________________________                                        .Iadd.Mass Spectrum                                                           ______________________________________                                        Observed molecular ion 558.3249                                               Calculated for C.sub.32 H.sub.46 O.sub.8                                                              558.3271                                              ______________________________________                                        .Iaddend.

m/e (relative intensity) 558(26), 529(44), 463(30), 343(12), 305(14),259(14), 245(21), 195(90) and 193(100)

NMR (CDCl₃) δ 0.89 (s, 3H, CH₃), 1.05 (m, 6H, 2CH₃), 1.59 (s, 3H, CH₃),1.64 (s, 3H, CH₃), 1.98 (s, 3H, CH₃ CO), 2.14 (s, 3H, CH₃ CO), 2.44 (q,1H, CH₂ CO), 2.65 (q, 1H, CH₂ CO), 3.07 (m, 1H, bisallyl), 3.86 (q, 1H,CH--O), 4.07-4.29 (m, 3H, 3CH--O), 4.67 (q, 1H, CH--OAc), 5.06 (q, 1H,vinyl), 5.25 (d, 1H, vinyl), 5.32-5.52 (m, 4H, 3 vinyl and 1CH--OAc) and5.57 (d, 1H, vinyl).

We claim:
 1. A process for preparing the substance acid F (V) having thefollowing formula: ##STR6## which comprises the following steps: A.Reducing an alcoholic solution of keto ester S (III) wherein the ketoester S has the following formula: ##STR7## with an alkali metalborohydride to obtain a mixture of acid S methyl ester II and acid Fmethyl ester IV having the following formulas: ##STR8## B. Separatingacid S methyl ester II and acid F methyl ester IV by preparative thinlayer chromatography;C. Hydrolyzing acid F methyl ester IV to obtain thedesired acid F (V).
 2. A process according to claim 1 wherein in Step A,the reduction is conducted in methanol using sodium borohydride.
 3. Aprocess according to claim 1 wherein in Step C, the hydrolysis isconducted in methanol using sodium hydroxide.